CN101153278A - Method for producing recombined human granular leukocyte colony stimulating factor - Google Patents

Abstract

The present invention provides an encoding sequence of recombinant human granulocyte colony-stimulating factor (rhG-CSF) as well as a production method of the recombinant human granulocyte colony-stimulating factor, and expression vectors and host cells used for the production method. The rhG-CSF is expressed by adopting pET30a(+) and a high level expression strain is obtained; a higher protein expression rate is achieved by controlling ferment conditions; a further analysis shows that the expression form is an inclusion body expression. A set of purification methods of the recombinant human granulocyte colony-stimulating factor are obtained after a lot of explorations and studies on the purification of the recombinant human granulocyte colony-stimulating factor secreted and expressed. The protein of recombinant human granulocyte colony-stimulating factor with a high yield rate and a high purity can be obtained by adopting the present invention and can satisfy clinical requirements. The present invention can efficiently, conveniently obtain the pure recombinant human granulocyte colony-stimulating factor at lower cost.

Description

The production method of recombinant methionyl human G-CSF

Technical field

The present invention relates to the genetically engineered field.More specifically, the invention provides a kind of production method of High-efficient Production recombinant methionyl human G-CSF, comprise the structure of relevant recombinant methionyl human G-CSF encoding sequence, engineering bacteria, the expression and the purifying process of recombinant methionyl human G-CSF.

Background technology

Granulocyte colony-stimulating factor (G-CSF) is vascular endothelial cell, monocyte and inoblast synthetic glycoprotein.G-CSF can promote neutrophil leucocyte (ANC) maturation; Stimulate mature granulocyte to disengage from marrow; Strengthen neutrophil leucocyte chemotactic and phagocytic function, influence very little scavenger cell, megalokaryocyte.G-CSF mainly acts on propagation, differentiation and the activation of neutrophil series (lineage) hematopoietic cell.Stimulate the formation of neutrophil leucocyte colony in the marrow hemopoiesis progenitor cell at external G-CSF, prolong the survival time of ripe neutrophil leucocyte, the activation neutrophil leucocyte promotes its ADCC, the generation of superoxide anion and alkaline phosphatase synthetic.

Studies show that recently, separately G-CSF or form with collaborative propagation, the stem cell parent cell colony that can promote pluripotential hemopoietic stem cell of STEM CELL FACTOR (SCF) and body in granular leukocyte colony form the formation of unit (CFU-G).G-CSF also has the chemotaxis to human granulocyte, monocyte, inoblast, smooth muscle cell and myofibroblast.

The human G-CSF assignment of genes gene mapping is in 17q21-22.RhG-CSF has 4 kinds of different forms: filg rastim (Neupogen), nartograstim (Ro25-8315), lenograstim (Granuocyte), SD/O1 (filgrastimsustained duration).The above two are applied to clinical in the U.S., both use the back in Europe.Lenograstim is only glycosylated rhG-CSF.In the experiment in vitro, when pH and temperature change, glycosylation can strengthen the stability of GCSF and the Decomposition P1 of antiserum(antisera) enzyme.In fact, the avidity of lenograstim and acceptor is 3 times of filgrastim: the former stimulate neutrophil leucocyte clone ripe aspect required concentration be the latter's 1/16.

Recombinant methionyl human G-CSF rhG-CSF, can be used as at tumour patient put, oligoleukocythemia after the chemotherapy, the modern genetic engineering pharmaceutical grade protein of treatment usefulness.Particularly infect and the AIDS infection at septicemia of newborn, HIV, the application in autoimmunity granulocytopenia and the tumor chemoradiotherapy has positive effect.Therefore, the method for the new High-efficient Production recombinant methionyl human G-CSF of exploitation is necessary.

Human G-CSF maturation protein is made up of 174 amino acid, and theoretical molecular is 18.7kD.The present invention is according to the aminoacid sequence of human G-CSF, according to e. coli codon preferences principle, the complete sequence of synthetic recombinant methionyl human G-CSF (G-CSF) gene, it is cloned into plasmid pET-30 (+), be transformed into e. coli bl21 (DE3) then, obtain the G-CSF high expression engineering through expression screening.Expression product has carried out structural identification through methods such as Western-blotting, mass spectroscopy and the order-checkings of amino acid N end, for the mass preparation of recombinant methionyl human G-CSF (rhG-CSF) is laid a good foundation.

Summary of the invention

Purpose of the present invention just provides a kind of method of efficient and/or easy production recombinant methionyl human G-CSF.

Another object of the present invention just provides the encoding sequence and the expression vector and the engineering strain that are used for this method of the recombinant methionyl human G-CSF of optimization.

In a first aspect of the present invention, just provided a kind of nucleotide sequence of coding recombinant methionyl human G-CSF of optimization.Referring to Fig. 1.

In a second aspect of the present invention, a kind of expression vector is provided, contain the described nucleotide sequence of claim 1.

In another preference, described expression vector is pET30a (+)/rhG-CSF.

In a third aspect of the present invention, a kind of engineering cell is provided, it is characterized in that, contain the described expression vector of claim 2.

In another preference, described engineering cell is e. coli bl21 (DE3), is the genetic flaw bacterial strain of a plurality of proteolytic enzyme, and genotype is hsdSgal (a λ cIts857ind1Sam7nim5lacUV5-T gene), is fit to very much efficiently expressing of foreign gene.

In a fourth aspect of the present invention, a kind of method of producing recombinant methionyl human G-CSF is provided, the method comprising the steps of:

A) under the expression condition that is fit to, cultivate host cell as claimed in claim 4, thereby give expression to recombinant methionyl human G-CSF; Preferably, described host cell is Bacillus coli cells BL21 (DE3).

B) separation and purification goes out the recombinant methionyl human G-CSF of expression.

In another preference of the present invention, rhG-CSF exists with the inclusion body form in intestinal bacteria, select the expression engineering bacteria that a strain is proved conclusively, cultivate and with 1mM IPTG abduction delivering with the LB substratum, collect thalline, through purification steps such as the broken bacterium of squeezing, inclusion body sex change, renaturation, cation-exchange chromatographies, end product purity reaches more than 90%.

Description of drawings

The homology of theoretical sequence of Fig. 1 .rhG-CSF and rhG-CSF majorizing sequence relatively.Query: rhG-CSF gene order originally; Sbjct: the rhG-CSF gene order of optimization.

The structure route map of Fig. 2 .pET30a (+)/rhG-CSF expression plasmid.

Embodiment

The inventor is extensive studies by going deep into, by optimization design rhG-CSF gene coded sequence, rhG-CSF encoding sequence after optimizing is built into suitable expression vector, transformed host cell, obtain the high expression level bacterial strain, and by optimizing fermentation, purifying process, realized that rhG-CSF efficiently expresses, and the rhG-CSF that gives expression to keeps biologic activity.Finished the present invention on this basis.

The present invention is according to the rhG-CSF natural acid sequence, press codon-bias, under the condition that does not change aminoacid sequence, optimize its nucleotide sequence, the synthetic proteic target gene sequences of rhG-CSF of full gene through optimizing, with this gene clone in the pET3 (+) after the sequence verification, ordinary method with molecular cloning, construction of expression vector changes intestinal bacteria over to, goes out to express engineering cell by applying antibiotic-screening.The test tube screening obtains the high expression level engineering cell.

In an example of the present invention, made up the escherichia coli expression engineering cell of producing rhG-CSF, IPTG induces, and high-level inclusion body is expressed.

In another example of the present invention, the optimization of technology has by fermentation further improved the expression amount of rhG-CSF.

In another example of the present invention, because the albumen inclusion body is expressed, thalline obtains inclusion body through homogenate, washing, can obtain pure product 120mg/L again behind a series of purification steps.

Stoste adds suitable auxiliary material behind the purifying, makes the powder ampoule agent for injection of rhG-CSF.The preliminarily stabilised test shows that this powder injection is stable.

Pilot scale research shows that product manufacture is stable, and is simple to operate, and the cycle is short, and cost is low, and every liter of fermented liquid can obtain the pure product 120mg of rhG-CSF, is fit to industrialization production.

The invention has the advantages that:

What (1) select for use is e. coli bl21 (DE3) host cell, goes out the multiple copied transformant by applying antibiotic-screening, and then filters out the high expression level engineering cell.

(2) the rhG-CSF gene after the optimization is highly suitable in e. coli bl21 (DE3) host cell and expresses, and has the characteristics of high expression level, high stable.

Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, people such as Sambrook for example, molecular cloning: laboratory manual (New York:Cold SpringHarbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.

The structure of embodiment 1 expression plasmid and the acquisition of high expression engineering strain

According to the aminoacid sequence of human G-CSF, according to e. coli codon preferences principle, the complete sequence of synthetic recombinant methionyl human G-CSF (G-CSF) gene,

According to the human G-CSF natural acid sequence, according to the e. coli codon preferences, under the condition that does not change aminoacid sequence, the complete sequence of full gene synthesizing recombined human granulocyte colony-stimulating factor (rhG-CSF) gene, this full length gene 525bp.Majorizing sequence and original series relatively see Fig. 1.Be cloned among the plasmid pET-30 (+) it and sequence verification.The expression vector establishment method is seen Fig. 2.

The PCR that handles gene rhG-CSF with NdeI and EcoRI double digestion respectively reclaims product and plasmid pET-30a (+), and 37 ℃ of enzymes were cut 3 hours, reclaimed corresponding fragment and spent the night 16 ℃ of connections with the T4DNA ligase enzyme.Connect the product conversion and enter bacillus coli DH 5 alpha, select positive colony and carry out the plasmid enzyme restriction evaluation on the LB flat board that contains kantlex (50g/mL): extractive plasmid DNA was reacted 3 hours at 37 ℃ with restriction enzyme NdeI and EcoRI, obtain in pET-30a (+), having inserted recombinant plasmid pET-30a (+)/rhG-CSF of rhGCSF gene, make up circuit and see Fig. 1.

Obtain purpose rhG-CSF gene by pcr amplification, the PCR that handles gene rhG-CSF with Nde I and EcoR I double digestion respectively reclaims product and plasmid pET30a (+), and enzyme is cut the back corresponding fragment of recovery and spent the night 16 ℃ of connections with the T4DNA ligase enzyme.Connect product transformed into escherichia coli DH5 α, select resistance male clone on the LB flat board of kantlex containing,

Expression plasmid pET-30a (+)/rhG-CSF conversion that builds is entered e. coli bl21 (DE3) competent cell that has prepared, and coating contains the LB flat board of kantlex (30g/mL), is transforming picking mono-clonal on the flat board then.With the alkaline lysis extracting and detect plasmid.The extracting plasmid DNA, and carry out plasmid enzyme restriction and identify.Obtain in pET30a (+), having inserted recombinant plasmid pET30a (+)/rhG-CSF of rhG-CSF gene.

Determining of the expression of embodiment 2 recombinant methionyl human G-CSFs and expression-form

Select expression engineering bacteria BL21 (DE3)/pET30a (+)/rhG-CSF that a strain is proved conclusively, cultivate and use the IPTG abduction delivering with the LB substratum, whether the SDS-PAGE electrophoresis detection has the appearance of purpose band, and analyzes its expression amount.The bacterium liquid of getting after a part is fermented is centrifugal, is resuspended in pH 8.0,20mM Tris damping fluid, and carrying out ultrasonic bacteria breaking under ice bath, the ultrasonic cleer and peaceful ultrasound precipitation of centrifugal collection, the SDS-PAGE electrophoresis detection is determined its expression-form.Analyze its ultrasonic cleer and peaceful ultrasound precipitation, the target protein overwhelming majority shows that target protein is with the inclusion body formal representation in precipitation.

All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.

Sequence table

＜110〉Shanghai Xinshengyuan Medicine Research Co., Ltd.

Shanghai Xinshengyuan Biological Medical Co., Ltd.

＜120〉production method of recombinant methionyl human G-CSF

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＜223〉the recombinant methionyl human G-CSF gene of You Huaing

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Claims (4)

1. the production method of a recombinant methionyl human G-CSF (rhG-CSF) is characterized in that it comprises following steps:

A. obtain the dna sequence dna of the recombination of human macrophage colony stimulating factor of optimization;

B. make up suitable expression vector;

C. transformed host cell screens the engineering cell that obtains high expression level.

2. the method for claim 1 is characterized in that, the aminoacid sequence shown in the dna sequence encoding SEQ ID NO:2 of the recombinant methionyl human G-CSF (rhG-CSF) of the optimization described in the step (a).Preferably, this dna sequence dna is shown in SEQ ID NO:1.

3. the method for claim 1 is characterized in that, the expression vector described in the step (b) contains the described dna sequence dna of claim 1.Preferably, described expression vector is pET30a (+)/rhG-CSF.

4. the method for claim 1 is characterized in that, the engineering cell described in the step (c) contains the described expression vector of claim 3.Preferably, described engineering cell is intestinal bacteria.

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